Glial Cell Development and Function in Zebrafish

The zebrafish is a premier vertebrate model system that offers many experimental advantages for in vivo imaging and genetic studies. This review provides an overview of glial cell types in the central and peripheral nervous system of zebrafish. We highlight some recent work that exploited the strengths of the zebrafish system to increase the understanding of the role of Gpr126 in Schwann cell myelination and illuminate the mechanisms controlling oligodendrocyte development and myelination. We also summarize similarities and differences between zebrafish radial glia and mammalian astrocytes and consider the possibility that their distinct characteristics may represent extremes in a continuum of cell identity. Finally, we focus on the emergence of zebrafish as a model for elucidating the development and function of microglia. These recent studies have highlighted the power of the zebrafish system for analyzing important aspects of glial development and function.Following the pioneering work of George Streisinger in the early 1980s, the zebrafish has emerged as a premier vertebrate model system (Streisinger et al. 1981). A key strength of the zebrafish is that the embryos and early larvae are transparent, allowing exquisite cellular analysis of many dynamic processes, including cell migration, axonal pathfinding, and myelination, among many others (e.g., Gilmour et al. 2002; Lyons et al. 2005; Czopka et al. 2013). The zebrafish also has many advantages for large-scale genetic studies, including relatively small size and rapid development, high fecundity, and the ability to manipulate the ploidy of gametes and early embryos (Kimmel 1989). Through the 1980s and early 1990s, insightful studies of several interesting mutations elegantly exploited these experimental advantages (e.g., Kimmel et al. 1989; Ho and Kane 1990; Hatta et al. 1991; Grunwald and Eisen 2002), attracting many researchers from other fields to the zebrafish system. Following the explosion of interest in the zebrafish in the 1990s, advances in many areas have added to the strengths of the system, including large-scale screens that identified thousands of new mutations (Driever et al. 1996; Haffter et al. 1996), rapid transgenesis (Kawakami et al. 2004), new methods for imaging and tracking all cells during development (Huisken 2012), genetic mapping and sequencing to identify genes and mutated loci (Postlethwait et al. 1994; Howe et al. 2013), optogenetic methods to control neural activity (Portugues et al. 2013), the advent of targeted nucleases to create mutations in genes of interest (Huang et al. 2011; Sander et al. 2011; Bedell et al. 2012; Chang et al. 2013; Hwang et al. 2013), and small molecule screening approaches to isolate compounds with novel biological activities in vivo (Peterson and Fishman 2011).Many fundamental similarities in physiology and body plan unite the zebrafish and other vertebrates (Kimmel 1989). In addition, analysis of genes and genomes has revealed that sequence, expression, and function of many genes are conserved among zebrafish and other vertebrates (Postlethwait and Talbot 1997; Howe et al. 2013). Thus, insights from studies in zebrafish will apply broadly to other vertebrates, including humans. On the other hand, there are important genetic, genomic, and physiological differences among vertebrates. It is, therefore, important to keep possible differences in mind and to recognize that analyzing the diversity among different species may enhance overall understanding of important processes. For example, zebrafish and other teleosts have a much more extensive regenerative ability than mammals, so that studies of fin, heart, and spinal cord regeneration in zebrafish may suggest avenues toward new therapeutic approaches in humans (Gemberling et al. 2013; Becker and Becker 2014).In this review, we provide an overview of different types of glia in the zebrafish, with a focus on some recent studies that highlight the power of the zebrafish system to analyze different aspects of glial development and function.     

Origin and Evolution of the Self-Organizing Cytoskeleton in the Network of Eukaryotic Organelles

The eukaryotic cytoskeleton evolved from prokaryotic cytomotive filaments. Prokaryotic filament systems show bewildering structural and dynamic complexity and, in many aspects, prefigure the self-organizing properties of the eukaryotic cytoskeleton. Here, the dynamic properties of the prokaryotic and eukaryotic cytoskeleton are compared, and how these relate to function and evolution of organellar networks is discussed. The evolution of new aspects of filament dynamics in eukaryotes, including severing and branching, and the advent of molecular motors converted the eukaryotic cytoskeleton into a self-organizing “active gel,” the dynamics of which can only be described with computational models. Advances in modeling and comparative genomics hold promise of a better understanding of the evolution of the self-organizing cytoskeleton in early eukaryotes, and its role in the evolution of novel eukaryotic functions, such as amoeboid motility, mitosis, and ciliary swimming.The eukaryotic cytoskeleton organizes space on the cellular scale and this organization influences almost every process in the cell. Organization depends on the mechanochemical properties of the cytoskeleton that dynamically maintain cell shape, position organelles, and macromolecules by trafficking, and drive locomotion via actin-rich cellular protrusions, ciliary beating, or ciliary gliding. The eukaryotic cytoskeleton is best described as an “active gel,” a cross-linked network of polymers (gel) in which many of the links are active motors that can move the polymers relative to each other (Karsenti et al. 2006). Because prokaryotes have only cytoskeletal polymers but lack motor proteins, this “active gel” property clearly sets the eukaryotic cytoskeleton apart from prokaryotic filament systems.Prokaryotes contain elaborate systems of several cytomotive filaments (Löwe and Amos 2009) that share many structural and dynamic features with eukaryotic actin filaments and microtubules (Löwe and Amos 1998; van den Ent et al. 2001). Prokaryotic cytoskeletal filaments may trace back to the first cells and may have originated as higher-order assemblies of enzymes (Noree et al. 2010; Barry and Gitai 2011). These cytomotive filaments are required for the segregation of low copy number plasmids, cell rigidity and cell-wall synthesis, cell division, and occasionally the organization of membranous organelles (Komeili et al. 2006; Thanbichler and Shapiro 2008; Löwe and Amos 2009). These functions are performed by dynamic filament-forming systems that harness the energy from nucleotide hydrolysis to generate forces either via bending or polymerization (Löwe and Amos 2009; Pilhofer and Jensen 2013). Although the identification of actin and tubulin homologs in prokaryotes is a major breakthrough, we are far from understanding the origin of the structural and dynamic complexity of the eukaryotic cytoskeleton.Advances in genome sequencing and comparative genomics now allow a detailed reconstruction of the cytoskeletal components present in the last common ancestor of eukaryotes. These studies all point to an ancestrally complex cytoskeleton, with several families of motors (Wickstead and Gull 2007; Wickstead et al. 2010) and filament-associated proteins and other regulators in place (Jékely 2003; Richards and Cavalier-Smith 2005; Rivero and Cvrcková 2007; Chalkia et al. 2008; Eme et al. 2009; Fritz-Laylin et al. 2010; Eckert et al. 2011; Hammesfahr and Kollmar 2012). Genomic reconstructions and comparative cell biology of single-celled eukaryotes (Raikov 1994; Cavalier-Smith 2013) allow us to infer the cellular features of the ancestral eukaryote. These analyses indicate that amoeboid motility (Fritz-Laylin et al. 2010; although, see Cavalier-Smith 2013), cilia (Cavalier-Smith 2002; Mitchell 2004; Jékely and Arendt 2006; Satir et al. 2008), centrioles (Carvalho-Santos et al. 2010), phagocytosis (Cavalier-Smith 2002; Jékely 2007; Yutin et al. 2009), a midbody during cell division (Eme et al. 2009), mitosis (Raikov 1994), and meiosis (Ramesh et al. 2005) were all ancestral eukaryotic cellular features. The availability of functional information from organisms other than animals and yeasts (e.g., Chlamydomonas, Tetrahymena, Trypanosoma) also allow more reliable inferences about the ancestral functions of cytoskeletal components (i.e., not only their ancestral presence or absence) and their regulation (Demonchy et al. 2009; Lechtreck et al. 2009; Suryavanshi et al. 2010).The ancestral complexity of the cytoskeleton in eukaryotes leaves a huge gap between prokaryotes and the earliest eukaryote we can reconstruct (provided that our rooting of the tree is correct) (Cavalier-Smith 2013). Nevertheless, we can attempt to infer the series of events that happened along the stem lineage, leading to the last common ancestor of eukaryotes. Meaningful answers will require the use of a combination of gene family history reconstructions (Wickstead and Gull 2007; Wickstead et al. 2010), transition analyses (Cavalier-Smith 2002), and computer simulations relevant to cell evolution (Jékely 2008).     

Cadherin-mediated Intercellular Adhesion and Signaling Cascades Involving Small GTPases

Epithelia form physical barriers that separate the internal milieu of the body from its external environment. The biogenesis of functional epithelia requires the precise coordination of many cellular processes. One of the key events in epithelial biogenesis is the establishment of cadherin-dependent cell–cell contacts, which initiate morphological changes and the formation of other adhesive structures. Cadherin-mediated adhesions generate intracellular signals that control cytoskeletal reorganization, polarity, and vesicle trafficking. Among such signaling pathways, those involving small GTPases play critical roles in epithelial biogenesis. Assembly of E-cadherin activates several small GTPases and, in turn, the activated small GTPases control the effects of E-cadherin-mediated adhesions on epithelial biogenesis. Here, we focus on small GTPase signaling at E-cadherin-mediated epithelial junctions.Cell–cell adhesions are involved in a diverse range of physiological processes, including morphological changes during tissue development, cell scattering, wound healing, and synaptogenesis (Adams and Nelson 1998; Gumbiner 2000; Halbleib and Nelson 2006; Takeichi 1995; Tepass et al. 2000). In epithelial cells, cell–cell adhesions are classified into three kinds of adhesions: adherens junction, tight junction, and desmosome (for more details, see Meng and Takeichi 2009, Furuse 2009, and Delva et al. 2009, respectively). A key event in epithelial polarization and biogenesis is the establishment of cadherin-dependent cell–cell contacts. Cadherins belong to a large family of adhesion molecules that require Ca²⁺ for their homophilic interactions (Adams and Nelson 1998; Blanpain and Fuchs 2009; Gumbiner 2000; Hartsock and Nelson 2008; Takeichi 1995; Tepass et al. 2000). Cadherins form transinteraction on the surface of neighboring cells (for details, see Shapiro and Weis 2009). For the development of strong and rigid adhesions, cadherins are clustered concomitantly with changes in the organization of the actin cytoskeleton (Tsukita et al. 1992). Classical cadherins are required, but not sufficient, to initiate cell–cell contacts, and other adhesion protein complexes subsequently assemble (for details, see Green et al. 2009). These complexes include the tight junction, which controls paracellular permeability, and desmosomes, which support the structural continuum of epithelial cells. A fundamental problem is to understand how these diverse cellular processes are regulated and coordinated. Intracellular signals, generated when cells attach with one another, mediate these complicated processes.Several signaling pathways upstream or downstream of cadherin-mediated cell–cell adhesions have been identified (Perez-Moreno et al. 2003) (see also McCrea et al. 2009). Among these pathways, small GTPases including the Rho and Ras family GTPases play critical roles in epithelial biogenesis and have been studied extensively. Many key morphological and functional changes are induced when these small GTPases act at epithelial junctions, where they mediate an interplay between cell–cell adhesion molecules and fundamental cellular processes including cytoskeletal activity, polarity, and vesicle trafficking. In addition to these small GTPases, Ca²⁺ signaling and phosphorylation of cadherin complexes also play pivotal roles in the formation and maintenance of cadherin-mediated adhesions. Here, we focus on signaling pathways involving the small GTPases in E-cadherin-mediated cell–cell adhesions. Other signaling pathways are described in recent reviews (Braga 2002; Fukata and Kaibuchi 2001; Goldstein and Macara 2007; McLachlan et al. 2007; Tsukita et al. 2008; Yap and Kovacs 2003; see also McCrea et al. 2009).     

Molecular Bases of Cell–Cell Junctions Stability and Dynamics

Epithelial cell–cell junctions are formed by apical adherens junctions (AJs), which are composed of cadherin adhesion molecules interacting in a dynamic way with the cortical actin cytoskeleton. Regulation of cell–cell junction stability and dynamics is crucial to maintain tissue integrity and allow tissue remodeling throughout development. Actin filament turnover and organization are tightly controlled together with myosin-II activity to produce mechanical forces that drive the assembly, maintenance, and remodeling of AJs. In this review, we will discuss these three distinct stages in the lifespan of cell–cell junctions, using several developmental contexts, which illustrate how mechanical forces are generated and transmitted at junctions, and how they impact on the integrity and the remodeling of cell–cell junctions.Cell–cell junction formation and remodeling occur repeatedly throughout development. Epithelial cells are linked by apical adherens junctions (AJs) that rely on the cadherin-catenin-actin module. Cadherins, of which epithelial E-cadherin (E-cad) is the most studied, are Ca²⁺-dependent transmembrane adhesion proteins forming homophilic and heterophilic bonds in trans between adjacent cells. Cadherins and the actin cytoskeleton are mutually interdependent (Jaffe et al. 1990; Matsuzaki et al. 1990; Hirano et al. 1992; Oyama et al. 1994; Angres et al. 1996; Orsulic and Peifer 1996; Adams et al. 1998; Zhang et al. 2005; Pilot et al. 2006). This has long been attributed to direct physical interaction of E-cad with β-catenin (β-cat) and of α-catenin (α-cat) with actin filaments (for reviews, see Gumbiner 2005; Leckband and Prakasam 2006; Pokutta and Weis 2007). Recently, biochemical and protein dynamics analyses have shown that such a link may not exist and that instead, a constant shuttling of α-cat between cadherin/β-cat complexes and actin may be key to explain the dynamic aspect of cell–cell adhesion (Drees et al. 2005; Yamada et al. 2005). Regardless of the exact nature of this link, several studies show that AJs are indeed physically attached to actin and that cadherins transmit cortical forces exerted by junctional acto-myosin networks (Costa et al. 1998; Sako et al. 1998; Pettitt et al. 2003; Dawes-Hoang et al. 2005; Cavey et al. 2008; Martin et al. 2008; Rauzi et al. 2008). In addition, physical association depends in part on α-cat (Cavey et al. 2008) and additional intermediates have been proposed to represent alternative missing links (Abe and Takeichi 2008) (reviewed in Gates and Peifer 2005; Weis and Nelson 2006). Although further work is needed to address the molecular nature of cadherin/actin dynamic interactions, association with actin is crucial all throughout the lifespan of AJs. In this article, we will review our current understanding of the molecular mechanisms at work during three different developmental stages of AJs biology: assembly, stabilization, and remodeling, with special emphasis on the mechanical forces controlling AJs integrity and development.     

Functional Differentiation of Adult-Born Neurons along the Septotemporal Axis of the Dentate Gyrus

Over the past several decades, the proliferation and integration of adult-born neurons into existing hippocampal circuitry has been implicated in a wide range of behaviors, including novelty recognition, pattern separation, spatial learning, anxiety behaviors, and antidepressant response. In this review, we suggest that the diversity in behavioral requirements for new neurons may be partly caused by separate functional roles of individual neurogenic niches. Growing evidence shows that the hippocampal formation can be compartmentalized not only along the classic trisynaptic circuit, but also along a longitudinal septotemporal axis. We suggest that subpopulations of hippocampal adult-born neurons may be specialized for distinct mnemonic- or mood-related behavioral tasks. We will examine the literature supporting a functional and anatomical dissociation of the hippocampus along the longitudinal axis and discuss techniques to functionally dissect the roles of adult-born hippocampal neurons in these distinct subregions.Since the presence of dividing cells in the mostly postmitotic adult brain was first described (Altman and Das 1965), the generation of new neurons in adulthood has been proposed to be involved in a variety of behaviors (Doetsch and Hen 2005; Becker and Wojtowicz 2007; Sahay and Hen 2007; Deng et al. 2010; Ming and Song 2011; Miller and Hen 2014). Adult neurogenesis in the healthy mammalian brain is consistently seen in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG). Recent studies have implicated hippocampal neurogenesis in learning- and memory-related tasks, such as contextual discrimination and spatial navigation and, specifically, in behavioral pattern separation (Clelland et al. 2009; Sahay et al. 2011; Nakashiba et al. 2012; Niibori et al. 2012; see also reviews in Deng et al. 2010; Ming and Song 2011; Marin-Burgin and Schinder 2012), but also in some behavioral effects of antidepressants (Santarelli et al. 2003; see also reviews in Sahay and Hen 2007; Kheirbek et al. 2012; Tanti and Belzung 2013). However, the exact role of adult hippocampal neurogenesis in some of these behaviors has been debated as some studies have shown no effects of altering adult neurogenesis on spatial navigation or antidepressant response. Proposed explanations have included differences in the behavioral tasks used to measure cognition or emotion, motivational state of subjects, species differences, or in how neurogenesis is defined, either as proliferation, survival, or differentiation (see reviews in Zhao et al. 2008; Aimone et al. 2011; Petrik et al. 2012b; Miller and Hen 2014).It must also be noted, however, that these hippocampal neurons are not born into a singular structure. Work in the past several decades has shown that the hippocampus can be divided, not only along the classic trisynaptic loop, but also longitudinally along a septotemporal axis. The septal (dorsal in rodents; posterior in primates) and temporal (ventral in rodents; anterior in primates) poles, as well as potential intermediate zones of the hippocampus, have different anatomic connections and electrophysiological properties, express a gradient of molecular markers, and play different functional roles, such as performance in spatial learning tasks and stress responses (see reviews in Moser and Moser 1998; Fanselow and Dong 2010). Consequently, adult-born neurons in the hippocampal DG may also be segregated along this longitudinal axis, and conflicting functional roles for neurogenesis may be a result of attempting to examine hippocampal neurogenesis as a unitary phenomenon. It is possible that there are intrinsic, cell-autonomous differences in adult-born neurons generated at opposite poles of the DG. An alternative, although not mutually exclusive, hypothesis is that progenitor cells are initially identical, but differentiate in a dissimilar manner as a result of integration into distinct network circuitry. We will, therefore, first discuss heterogeneity of the hippocampus along its longitudinal axis before reviewing differences in neurogenesis between the septal and temporal poles of the DG. As these topics have been reviewed extensively elsewhere (Moser and Moser 1998; Deng et al. 2010; Fanselow and Dong 2010; Koehl and Abrous 2011; Samuels and Hen 2011; Kheirbek et al. 2012; Petrik et al. 2012b), we will not try to exhaustively cover all the current literature. Rather, we attempt to gather key studies examining a septotemporal gradient of the hippocampus and hippocampal neurogenesis. We will then suggest possible approaches to examine neurogenesis in specific subregions of the hippocampal DG. Finally, a short section will examine segregation of the DG along its transverse axis.     

Biology of the TAM Receptors

The TAM receptors—Tyro3, Axl, and Mer—comprise a unique family of receptor tyrosine kinases, in that as a group they play no essential role in embryonic development. Instead, they function as homeostatic regulators in adult tissues and organ systems that are subject to continuous challenge and renewal throughout life. Their regulatory roles are prominent in the mature immune, reproductive, hematopoietic, vascular, and nervous systems. The TAMs and their ligands—Gas6 and Protein S—are essential for the efficient phagocytosis of apoptotic cells and membranes in these tissues; and in the immune system, they act as pleiotropic inhibitors of the innate inflammatory response to pathogens. Deficiencies in TAM signaling are thought to contribute to chronic inflammatory and autoimmune disease in humans, and aberrantly elevated TAM signaling is strongly associated with cancer progression, metastasis, and resistance to targeted therapies.The name of the TAM family is derived from the first letter of its three constituents—Tyro3, Axl, and Mer (Prasad et al. 2006). As detailed in Figure 1, members of this receptor tyrosine kinase (RTK) family were independently identified by several different groups and appear in the early literature under multiple alternative names. However, Tyro3, Axl, and Mer (officially c-Mer or MerTK for the protein, Mertk for the gene) have now been adopted as the NCBI designations. The TAMs were first grouped into a distinct RTK family (the Tyro3/7/12 cluster) in 1991, through PCR cloning of their kinase domains (Lai and Lemke 1991). The isolation of full-length cDNAs for Axl (O''Bryan et al. 1991), Mer (Graham et al. 1994), and Tyro3 (Lai et al. 1994) confirmed their segregation into a structurally distinctive family of orphan RTKs (Manning et al. 2002b). The two ligands that bind and activate the TAMs—Gas6 and Protein S (Pros1)—were identified shortly thereafter (Ohashi et al. 1995; Stitt et al. 1995; Mark et al. 1996; Nagata et al. 1996).Open in a separate windowFigure 1.TAM receptors and ligands. The TAM receptors (red) are Tyro3 (Lai and Lemke 1991; Lai et al. 1994)—also designated Brt (Fujimoto and Yamamoto 1994), Dtk (Crosier et al. 1994), Rse (Mark et al. 1994), Sky (Ohashi et al. 1994), and Tif (Dai et al. 1994); Axl (O''Bryan et al. 1991)—also designated Ark (Rescigno et al. 1991), Tyro7 (Lai and Lemke 1991), and Ufo (Janssen et al. 1991); and Mer (Graham et al. 1994)—also designated Eyk (Jia and Hanafusa 1994), Nyk (Ling and Kung 1995), and Tyro12 (Lai and Lemke 1991). The TAMs are widely expressed by cells of the mature immune, nervous, vascular, and reproductive systems. The TAM ligands (blue) are Gas6 and Protein S (Pros1). The carboxy-terminal SHBG domains of the ligands bind to the immunoglobulin (Ig) domains of the receptors, induce dimerization, and activate the TAM tyrosine kinases. When γ-carboxylated in a vitamin-K-dependent reaction, the amino-terminal Gla domains of the dimeric ligands bind to the phospholipid phosphatidylserine expressed on the surface on an apposed apoptotic cell or enveloped virus. See text for details. (From Lemke and Burstyn-Cohen 2010; adapted, with permission, from the authors.)Subsequent progress on elucidating the biological roles of the TAM receptors was considerably slower and ultimately required the derivation of mouse loss-of-function mutants (Camenisch et al. 1999; Lu et al. 1999). The fact that Tyro3^−/−, Axl^−/−, and Mer^−/− mice are all viable and fertile permitted the generation of a complete TAM mutant series that included all possible double mutants and even triple mutants that lack all three receptors (Lu et al. 1999). Remarkably, these Tyro3^−/−Axl^−/−Mer^−/− triple knockouts (TAM TKOs) are viable, and for the first 2–3 wk after birth, superficially indistinguishable from their wild-type counterparts (Lu et al. 1999). Because many RTKs play essential roles in embryonic development, even single loss-of-function mutations in RTK genes often result in an embryonic-lethal phenotype (Gassmann et al. 1995; Lee et al. 1995; Soriano 1997; Arman et al. 1998). The postnatal viability of mice in which an entire RTK family is ablated completely—the TAM TKOs can survive for more than a year (Lu et al. 1999)—is therefore highly unusual. Their viability notwithstanding, the TAM mutants go on to develop a plethora of phenotypes, some of them debilitating (Camenisch et al. 1999; Lu et al. 1999; Lu and Lemke 2001; Scott et al. 2001; Duncan et al. 2003; Prasad et al. 2006). Almost without exception, these phenotypes are degenerative in nature and reflect the loss of TAM signaling activities in adult tissues that are subject to regular challenge, renewal, and remodeling. These activities are the subject of this review.     

Self-avoidance and Tiling: Mechanisms of Dendrite and Axon Spacing

The spatial pattern of branches within axonal or dendritic arbors and the relative arrangement of neighboring arbors with respect to one another impact a neuron''s potential connectivity. Although arbors can adopt diverse branching patterns to suit their functions, evenly spread branches that avoid clumping or overlap are a common feature of many axonal and dendritic arbors. The degree of overlap between neighboring arbors innervating a surface is also characteristic within particular neuron types. The arbors of some populations of neurons innervate a target with a comprehensive and nonoverlapping “tiled” arrangement, whereas those of others show substantial territory overlap. This review focuses on cellular and molecular studies that have provided insight into the regulation of spatial arrangements of neurite branches within and between arbors. These studies have revealed principles that govern arbor arrangements in dendrites and axons in both vertebrates and invertebrates. Diverse molecular mechanisms controlling the spatial patterning of sister branches and neighboring arbors have begun to be elucidated.Axonal and dendritic arbors adopt complex and morphologically diverse shapes that influence neural connectivity and information processing. In this article we review anatomical and molecular studies that elucidate how the arrangements of branches within neuronal arbors are established during development (isoneuronal spacing) and how the relative spacing of arbors is determined when multiple neurons together innervate a defined territory (heteroneuronal spacing). Together these mechanisms ensure that arbors achieve functionally appropriate coverage of input or output territories.Isoneuronal and heteroneuronal processes display a variety of spacing arrangements, suggesting a diversity of underlying molecular mechanisms. Self-avoidance can occur between branches that arise from a single soma (Yau 1976; Kramer and Kuwada 1983; Kramer and Stent 1985), implying that neurons are able to discriminate “self,” which they avoid, from “nonself” arbors, with which they coexist (Kramer and Kuwada 1983). Similarly, arbors from different cells that share the same function and together innervate a defined territory can create a pattern of minimally overlapping neighboring dendritic or axonal fields, known as tiling. Such spacing mechanisms ensure that arbors maximize their spread across a territory while minimizing the redundancy with which the territory is innervated. In contrast, adhesive interactions between arbors can operate to maintain coherence of dendrites at specific targets (Zhu and Luo 2004), or to bundle functionally similar processes and possibly coordinate their activity (Campbell et al. 2009). Understanding how processes are patterned relative to one another can help to uncover the functional logic of neural circuit organization.Here we focus primarily on mechanisms of isoneuronal and heteroneuronal avoidance that result in complete and nonredundant innervation of sensory or synaptic space. Such mechanisms have been studied extensively in systems where neuronal arbors innervate a two-dimensional plane, such as the retina or body wall (Wassle et al. 1981; Perry and Linden 1982; Hitchcock 1989; Lin and Masland 2004; Fuerst et al. 2009; Kramer and Stent 1985; Grueber et al. 2003; Sugimura et al. 2003; Sagasti et al. 2005). However, the principles regulating process spacing in these regions likely also apply in three dimensions, most prominently where processes are segregated into nonoverlapping domains or columns (Huckfeldt et al. 2009). It is also notable that nonneuronal cell types might similarly engage in self-avoidance and form tiling arrangements, including leech comb cells (Jellies and Kristan 1991) and mammalian astrocytes (Bushong et al. 2002; Ogata and Kosaka 2002; Livet et al. 2007). Elucidating the mechanisms of process spacing during development is therefore relevant for understanding principles of tissue organization inside and outside of the nervous system.     

Retrograde Traffic from the Golgi to the Endoplasmic Reticulum

Proteins to be secreted are transported from the endoplasmic reticulum (ER) to the Golgi apparatus. The transport of these proteins requires the localization and activity of proteins that create ER exit sites, coat proteins to collect cargo and to reshape the membrane into a transport container, and address labels—SNARE proteins—to target the vesicles specifically to the Golgi apparatus. In addition some proteins may need export chaperones or export receptors to enable their exit into transport vesicles. ER export factors, SNAREs, and misfolded Golgi-resident proteins must all be retrieved from the Golgi to the ER again. This retrieval is also part of the organellar homeostasis pathway essential to maintaining the identity of the ER and of the Golgi apparatus. In this review, I will discuss the different processes in retrograde transport from the Golgi to the ER and highlight the mechanistic insights we have obtained in the last couple of years.Proteins that are exposed at the plasma membrane or populate a membrane-bounded organelle are synthesized into the endoplasmic reticulum (ER). In the ER, the folding of these proteins takes place and posttranslational modifications such as N-glycosylation and disulfide bridge formation occur. Upon adopting a suitable, often correct, conformation, proteins destined to locations beyond the ER are concentrated at so-called ER exit sites (ERES) and incorporated into nascent COPII-coated vesicles. These COPII vesicles eventually bud off the ER membrane and are transported to the Golgi (in yeast, Drosophila, and C. elegans) or the ER-Golgi intermediate compartment (in mammalian cells) (Schweizer et al. 1990; Kondylis and Rabouille 2003; Spang 2009; Witte et al. 2011).It is assumed that the vesicle coat is at least partially destabilized through the hydrolysis of GTP by the small GTPase Sar1 (Oka and Nakano 1994; Springer et al. 1999). However, some of the destabilized coat components have to stay on the vesicle until it has reached the Golgi apparatus because coat components participate in the recognition and the tethering process (Barlowe 1997; Cai et al. 2007; Lord et al. 2011; Zong et al. 2012). Subsequently, SNARE proteins on the vesicles (v-SNAREs) zipper up with cognate SNAREs on the Golgi (target SNAREs, t-SNAREs) to drive membrane fusion (Hay et al. 1998; Cao and Barlowe 2000; Parlati et al. 2002). The content of the ER-derived COPII vesicles is thereby released into the lumen of the cis-cisterna of the Golgi apparatus. Most proteins will continue their journey through the Golgi apparatus and encounter further modifications such as extension of the glycosylation tree or lipidation. However, some proteins, especially those involved in the fusion process, i.e., the v-SNAREs or proteins that act as export factors of the ER, such as Vma21, which is essential for export of the correctly folded and assembled V0 sector of the V-ATPase, need to be recycled back to the ER for another round of transport (Ballensiefen et al. 1998; Malkus et al. 2004). Moreover, cis-Golgi proteins are returned to the ER for quality/functional control (Todorow et al. 2000; Sato et al. 2004; Valkova et al. 2011). Finally, some ER-resident proteins, such as the ER Hsp70 chaperone BiP/Kar2, can escape the ER, but are captured at the cis-Golgi by the H/KDEL receptor Erd2 and returned to the ER (Lewis et al. 1990; Semenza et al. 1990; Aoe et al. 1997).Unfortunately, the retrograde transport route is also hijacked by toxins. For example, endocytosed cholera toxin subunit A contains a KDEL sequence and can thereby exploit the system to access the ER (Majoul et al. 1996, 1998). From there, it is retro-translocated into the cytoplasm where it can exert its detrimental function.     

Replicating Damaged DNA in Eukaryotes

DNA damage is one of many possible perturbations that challenge the mechanisms that preserve genetic stability during the copying of the eukaryotic genome in S phase. This short review provides, in the first part, a general introduction to the topic and an overview of checkpoint responses. In the second part, the mechanisms of error-free tolerance in response to fork-arresting DNA damage will be discussed in some detail.Before eukaryotic cells divide, the successful completion of DNA replication during S phase is essential to preserve genomic integrity from one generation to the next. During this process, the replication apparatus traverses in the form of bidirectionally moving forks to synthesize new daughter strands. Cells use several means to ensure faithful copying of the parental strands—first, by means of regulatory mechanisms a correctly coordinated replication apparatus is established, and second, a high degree of fidelity during DNA synthesis is maintained by replicative polymerases (Kunkel and Bebenek 2000; Reha-Krantz 2010). However, under several stressful circumstances, endogenously or exogenously induced, the replication apparatus can stall (Tourriere and Pasero 2007). Mostly, structural deformations in the form of lesions or special template-specific features arrest the replication process, activate checkpoint pathways and set in motion repair or tolerance mechanisms to counter the stalling (Branzei and Foiani 2009; Zegerman and Diffley 2009). Basic replication mechanism, its regulatory pathways and means to tolerate DNA damage are largely conserved across eukaryotic species (Branzei and Foiani 2010; Yao and O’Donnell 2010). Understanding the mechanisms involved may enable therapeutic intervention to several human conditions arising from an incomplete replication or from the inability to tolerate perturbations (Ciccia et al. 2009; Preston et al. 2010; Abbas et al. 2013). Enhanced replication stress has also been commonly identified in precancerous lesions, and the inactivation of checkpoint responses coping with this presumably oncogene-induced condition is considered necessary to establish the fully malignant phenotype (Bartkova et al. 2005; Negrini et al. 2010).It is not possible to treat this topic in a comprehensive manner in the allotted space; the reader is referred to excellent recent reviews for more details (Branzei and Foiani 2010; Jones and Petermann 2012). We will attempt to provide an overview of the various strategies that a eukaryotic cell invokes to avoid problems caused by replication stress related to DNA damage and, if problems arise, to tolerate damage without endangering the entire process of genome duplication. In this context, we will only give a brief outline of checkpoint responses that are discussed in more detail in Sirbu and Cortez (2013) and Marechal and Zou (2013). Also, a detailed discussion of translesion synthesis can be reviewed in Sale (2013).     

Unconventional Functions for Clathrin,ESCRTs, and Other Endocytic Regulators in the Cytoskeleton,Cell Cycle,Nucleus, and Beyond: Links to Human Disease

The roles of clathrin, its regulators, and the ESCRT (endosomal sorting complex required for transport) proteins are well defined in endocytosis. These proteins can also participate in intracellular pathways that are independent of endocytosis and even independent of the membrane trafficking function of these proteins. These nonendocytic functions involve unconventional biochemical interactions for some endocytic regulators, but can also exploit known interactions for nonendocytic functions. The molecular basis for the involvement of endocytic regulators in unconventional functions that influence the cytoskeleton, cell cycle, signaling, and gene regulation are described here. Through these additional functions, endocytic regulators participate in pathways that affect infection, glucose metabolism, development, and cellular transformation, expanding their significance in human health and disease.The discovery and characterization of clathrin (Pearse 1975) initiated molecular definition of the many endocytosis regulators described in this collection, which mediate the clathrin-dependent and -independent pathways for membrane internalization (see Kirchhausen et al. 2014; Mayor et al. 2014; Merrifield and Kaksonen 2014). In accompanying reviews, we have seen how these endocytic pathways influence nutrition and metabolism (see Antonescu et al. 2014), signal transduction (see Bökel and Brand 2014; Di Fiore and von Zastrow 2014), neuronal function (see Morgan et al. 2013; Cosker and Segal 2014), infection and immunity (see ten Broeke et al. 2013; Cossart and Helenius 2014), tissue polarity and development (see Eaton and Martin-Belmonte 2014; Gonzalez-Gaitan and Jülicher 2014), and migration and metastasis (see Mellman and Yarden 2013). Recently, it has been established that some endocytic regulators have molecular properties that expand their functions beyond endocytosis. These include molecular interactions that affect the microtubule and actin cytoskeletons, nuclear translocation that influences gene regulation, and the formation of membrane-associated scaffolds that serve as signaling and sorting platforms. Through these diverse nonendocytic functions, endocytosis regulators play additional roles in cell division, pathogen infection, cell adhesion, and oncogenesis. In this article, we review the nonconventional behavior of endocytic regulators, first discussing the molecular properties that enable their moonlighting functions and then discussing the cellular processes and disease states that are influenced by these functions.     

Quality Control of Mitochondrial Proteostasis

A decline in mitochondrial activity has been associated with aging and is a hallmark of many neurological diseases. Surveillance mechanisms acting at the molecular, organellar, and cellular level monitor mitochondrial integrity and ensure the maintenance of mitochondrial proteostasis. Here we will review the central role of mitochondrial chaperones and proteases, the cytosolic ubiquitin-proteasome system, and the mitochondrial unfolded response in this interconnected quality control network, highlighting the dual function of some proteases in protein quality control within the organelle and for the regulation of mitochondrial fusion and mitophagy.In all cellular compartments, correct protein folding is critical to maintain cellular homeostasis. In cases where proteins become misfolded or damaged, it is imperative that they are turned over and removed to prevent the formation of toxic folding intermediates or the accumulation of aggregates to levels that can be deleterious for the cell. Several neurodegenerative diseases share a common pathogenic mechanism, which involves the formation of fibrillar aggregates of a particular protein that can accumulate in the cytosol, the nucleus, or the mitochondria. Examples of this include accumulation of the amyloid-β peptide in Alzheimer’s disease (Kayed et al. 2003; Tanzi and Bertram 2005), accumulation of α-synuclein in Parkinson’s disease (Spillantini et al. 1997; Zarranz et al. 2004), and aggregation of a mutant form of the huntingtin protein caused by extended polyglutamine stretches in Huntington’s disease (DiFiglia et al. 1997). Although the exact mechanism of pathogenesis for these diseases remains unresolved, mitochondrial dysfunction is implicated in their progression, which may in turn be responsible for the loss of neurological cell populations because of their sensitivity and requirement for functional mitochondria (Rodolfo et al. 2010).The evolution of mitochondria began approximately 1.5 billion years ago after an α-proteobacterium was engulfed by a preeukaryotic cell (Gray et al. 1999). Since that time, mitochondria have retained two phospholipid bilayers that segregate two aqueous compartments, the mitochondrial intermembrane space (IMS) and the mitochondrial matrix (Palade 1953). Mitochondria are found in essentially all eukaryotic cells and play integral roles in a number of the cell''s metabolic pathways. For example, mitochondria are the key players in cellular ATP production through an elaborate respiratory chain network found in the organelles inner membrane (IM) (Mitchell 1961; Leonard and Schapira 2000). Mitochondria are also required for the β-oxidation of fatty acids, Fe-S biosynthesis, and Ca²⁺ homeostasis (Pinton et al. 1998; Rizzuto et al. 2000; Lill 2009; Modre-Osprian et al. 2009). Moreover, mitochondria are key regulators of programmed cell death and they participate in developmental processes as well as aging (Singh 2004; Green 2005).In contrast to early depictions of mitochondria as singular kidney bean shaped entities, it is now well established that mitochondria form elaborate, reticular networks in many tissues (Bereiter-Hahn 1990). The ability of mitochondria to form such networks arises from two major factors: (1) Specialized machineries in the mitochondrial outer membrane (OM) and the IM allow mitochondria to fuse and divide and (2) mitochondria are able to be shuttled along cytoskeletal elements (Anesti and Scorrano 2006; Hoppins et al. 2007). This plasticity of mitochondria ensures that they are able to respond to different cellular cues, which is potentially important for their numerous functions. In different cell types, mitochondria adopt varying morphologies (Kuznetsov et al. 2009). For example, in cultured fibroblasts mitochondria form extensive reticular networks, whereas in neuronal cells, mitochondria can be found enriched at areas of high-energy demand, including presynaptic termini, axon initial segments, and growth cones. Furthermore, in muscle cells, mitochondria adopt a very uniform intermyofibrillar conformation (Vendelin et al. 2005). The dynamic nature of mitochondria provides an explanation as to how they adopt varying organizations in different cell populations. The importance of mitochondrial networks is highlighted by the fact that mutations in components involved in maintaining mitochondrial dynamics results in neurodegenerative diseases (Chan 2006; Olichon et al. 2006; Knott et al. 2008; Martinelli and Rugarli 2010; Winklhofer and Haass 2010).     

Architecture of the Mammalian Golgi

Since its first visualization in 1898, the Golgi has been a topic of intense morphological research. A typical mammalian Golgi consists of a pile of stapled cisternae, the Golgi stack, which is a key station for modification of newly synthesized proteins and lipids. Distinct stacks are interconnected by tubules to form the Golgi ribbon. At the entrance site of the Golgi, the cis-Golgi, vesicular tubular clusters (VTCs) form the intermediate between the endoplasmic reticulum and the Golgi stack. At the exit site of the Golgi, the trans-Golgi, the trans-Golgi network (TGN) is the major site of sorting proteins to distinct cellular locations. Golgi functioning can only be understood in light of its complex architecture, as was revealed by a range of distinct electron microscopy (EM) approaches. In this article, a general concept of mammalian Golgi architecture, including VTCs and the TGN, is described.In 1898 Camillo Golgi was the first to visualize, describe, and ultimately name the Golgi complex. Using a histochemical impregnation method causing the reduction and deposition of silver, he defined the Golgi in neuronal cells as a reticular apparatus stained by the “black reaction” (Golgi 1898). In the 1950s, the first ultrastructural images of the Golgi were revealed using the then newly developed electron microscope (EM) (Dalton 1954; Farquhar and Rinehart 1954; Sjostrand and Hanzon 1954; Dalton and Felix 1956), reviewed by Farquhar and Palade (1981). In 1961, the thiamine pyrophosphatase reaction developed by Novikoff and Goldfischer allowed cytochemical labeling of Golgi membranes, which revealed the ubiquitous cellular distribution of this organelle (Novikoff and Goldfischer 1961). In the many years of ultrastructural research that have followed, the visualization of the Golgi has gone hand-in-hand with the developing EM techniques.The intriguing structural complexity of the Golgi has made it one of the most photographed organelles in the cell. However, a full understanding of Golgi architecture is hard to deduce from the ultrathin (70–100 nm) sections used in standard transmission EM preparations. Rambourg and Clermont (1974) were the first to investigate the Golgi in three dimensions (3D), using stereoscopy (Rambourg 1974). In this approach a “thick” (150–200 nm), EM section is photographed at two distinct angles, after which the pairs of photographs are viewed with a stereoscope. Over the years, stereoscopy was applied to a variety of cells and has greatly contributed to our current understanding of Golgi architecture (Lindsey and Ellisman 1985; Rambourg and Clermont 1990; Clermont et al. 1994; Clermont et al. 1995). An alternative approach to study 3D structure is serial sectioning, by which a series of adjacent (serial) thin sections are collected. The Golgi can be followed throughout these sections and be constructed into a 3D model (Beams and Kessel 1968; Dylewski et al. 1984; Rambourg and Clermont 1990). In the nineties, 3D-EM was boosted by the introduction of high-voltage, dual axis 3D electron tomography (Ladinsky et al. 1999; Koster and Klumperman 2003; Marsh 2005; Marsh 2007; Noske et al. 2008), which allows the analysis of sections of up to 3–4 µm with a 4–6 nm resolution in the z-axis. The sections are photographed in a tilt series of different angles, which are reconstructed into a 3D tomogram that allows one to “look beyond” a given structure and reveals how it relates to other cellular compartments.Membranes with a similar appearance can differ in protein content and function. These differences are revealed by protein localization techniques. Therefore, in addition to the “classical” EM techniques providing ultrastructural details, EM methods that determine protein localization within the context of the cellular morphology have been crucial to further our understanding on the functional organization of the Golgi. For example, by enzyme-activity-based cytochemical staining the cis-to-trans-polarity in the distribution of Golgi glycosylation enzymes was discovered, reviewed by Farquhar and Palade (1981), which was key to understanding the functional organization of the Golgi stack in protein and lipid glycosylation. With the development of immunoEM methods, using antibodies, the need for enzyme activity for protein localization was overcome. This paved the way for the localization of a wide variety of proteins, such as the cytoplasmic coat complexes associated with the Golgi (Rabouille and Klumperman 2005).A logical next step in EM-based imaging of the Golgi would be to combine protein localization with 3D imaging, but this is technically challenging. A number of protocols enabling protein localization in 3D have recently been described (Trucco et al. 2004; Grabenbauer et al. 2005; Gaietta et al. 2006; Zeuschner et al. 2006; Meiblitzer-Ruppitsch et al. 2008), but these have only been applied in a limited manner to Golgi studies. Another approach that holds great potential for Golgi research is correlative microscopy (CLEM). Live cell imaging of fluorescent proteins has revolutionized cell biology by the real time visualization of dynamic events. However, live cell imaging does not reveal membrane complexity. By CLEM, live cells are first viewed by light microscopy and then prepared for EM (Mironov et al. 2008; van Rijnsoever et al. 2008). When coupled with the recent introduction of super resolution light microscopy techniques for real time imaging, the combination with EM for direct correlation with ultrastructural resolution has great potential (Hell 2009; Lippincott-Schwartz and Manley 2009).The 100th anniversary of the discovery of the Golgi, in 1998, triggered a wave of reviews on this organelle, including those focusing on Golgi architecture (Rambourg 1997; Farquhar and Palade 1998). More recent reviews that describe Golgi structure in great detail are provided by Marsh (2005) and Hua (2009). In this article, the most recent insights in mammalian Golgi architecture as revealed by distinct EM approaches are integrated into a general concept.     

Myelin under construction -- teamwork required

Myelinating glial cells synthesize specialized myelin proteins and deposit them in the growing myelin sheath that enwraps axons multiple times. How do axons and myelinating glial cells coordinate this spectacular cell–cell interaction? In this issue, Trajkovic et al. (p. 937) show that neuronal signaling regulates cell surface expression of the myelin proteolipid protein in cultured oligodendrocytes in unexpected ways that may also contribute to myelination in situ.Myelination is a stunning example of how multiple cells cooperate to build a complex structure. Understanding how myelinating glia and neurons work together to achieve this feat is thus a challenging and important problem. Trajkovic et al. (p. 937) investigate the regulation of the trafficking of a major myelin protein, proteolipid protein (PLP), to the plasma membrane (PM) of cultured oligodendrocytes (OLs). When initially expressed in cultured OLs, PLP resides in a compartment with characteristics of a late endosome/lysosome (LE/L). Co-culture with neurons leads to an increase of PLP on the PM and a disappearance from the LE/L. This increased surface expression of PLP is due to at least two distinct mechanisms: a decrease in PLP endocytosis from the PM and an increase in exocytosis from the LE/L. The relative contributions of these two mechanisms (and possibly additional ones?) remain open questions for the future.The cells that produce myelin are highly specialized glial cells, Schwann cells in the peripheral nervous system (PNS) and OLs in the central nervous system (CNS). Myelin consists of many wrappings of glial cell membrane around the axon with little or no cytoplasm left between adjacent wraps. This compact myelin region insulates the axon from the extracellular medium and allows saltatory conduction along axons. Each successive myelin wrap creates at its lateral margins a membrane loop containing some cytoplasm. These so-called paranodal loops make up part of the noncompact myelin. Each paranodal loop forms a specialized cell junction with the axon, the axoglial apparatus. The paranodal loops, in turn, flank Nodes of Ranvier, gaps in the myelin where voltage-gated sodium channels cluster and regenerate the action potential (for review see Sherman and Brophy, 2005).Myelination is a supreme example of differential protein distribution. During myelination, glia elaborate distinct domains (such as soma and compact and noncompact myelin) with distinct lipids and protein components. At the same time, axonal membrane proteins also accumulate in distinct regions, such that the Node of Ranvier contains different proteins than the paranodal region (underlying the paranodal loops) or the juxtaparanode (flanking the paranode). Much work on who signaled whom, when, and why, revealed that neurons and myelinating glia communicate with each other bidirectionally in multiple ways to orchestrate myelination (Sherman and Brophy 2005). For instance, glial cells signal to neurons to influence axonal diameter, neurofilament spacing, and phosphorylation (Hsieh et al., 1994). Additionally, nodal, paranodal, and juxtaparanodal domains on axons form as a result of interactions with glial cells. Mutations in genes encoding paranodal proteins lead to aberrant paranodal loops and mislocalization of paranodal and juxtaparanodal components in the axon (for review see Poliak and Peles, 2003; Salzer, 2003). Somewhat surprisingly, nodal proteins still cluster in these mice, leading to the suggestion that nodal assembly might be intrinsic to axons or (in the CNS) driven by diffusible glial-derived factors (Kaplan et al., 1997). New work argues that glial cell processes which contact the node itself could direct nodal assembly. In the PNS, the node is contacted directly by microvilli of the myelinating Schwann cell. Mice lacking Schwann cell dystroglycan or laminin have aberrant microvilli and poorly clustered voltage-gated sodium channels (Saito et al., 2003; Occhi et al., 2005). Gliomedin, identified by the Peles lab, is expressed in Schwann cell microvilli and required for clustering of nodal axonal components (Eshed et al., 2005). In the CNS, Colman''s group localized the outgrowth-inhibitory molecule Omgp to distinct glial cells that can encircle nodes (Huang et al., 2005). Omgp knock-out mice show wider and disorganized nodes as well as aberrant sprouting of branches from nodes. These findings highlight the importance of node-encircling glial cells for organizing the axon.Do neurons in turn give instructions to glial cells? Oligodendrocyte precursor cells (OPCs) in the CNS migrate into developing white matter where they differentiate into postmitotic OLs and produce the myelin sheath. The differentiation of OPCs in terms of changes in gene expression and in morphology has been studied extensively in vitro and in vivo (for reviews see Pfeiffer et al., 1993; Barres and Raff, 1999). Because OPCs differentiate normally in axon-free culture and express myelin components, a role for neurons was not immediately apparent. In vivo, on the other hand, few OLs develop after transection of the optic nerve and subsequently, axons were shown to be required for survival and differentiation of OLs (Barres and Raff, 1999). OPCs and newly born OLs require astrocyte-derived factors such as PDGF, but OLs become dependent on axonal signals later. Axonal signaling to OLs occurs on at least two levels (Barres and Raff, 1999; Coman et al., 2005). Electrical activity (mediated by extrasynaptic release of adenosine [Stevens et al., 2002]) is required for proliferation of OPCs. Additionally, contact-mediated neuronal signals play important roles in OPC and Schwann cell differentiation and myelination (Corfas et al., 2004). Salzer and colleagues recently showed that the levels of neuregulin 1 type III expressed on axons determine the ensheathment fate of axons in the PNS (Taveggia et al., 2005).Compact myelin has a very specific composition of 70% lipids by dry weight (mostly composed of galactoceramide and cerebroside) with 80% of the protein mass comprised of only two proteins, myelin basic protein MBP and proteolipid protein PLP/DM20 (for review see Kramer et al., 2001). Studies have therefore focused on how OLs synthesize MBP and PLP and incorporate them into the growing myelin sheath. MBP is synthesized on free ribosomes, but its mRNA is localized to the myelin sheath (Colman et al., 1982). PLP on the other hand is a membrane-spanning protein that traverses the ER and Golgi. The role for axonal signaling for production of the myelin sheath is not well understood. For instance, OPCs in culture undergo differentiation and start to synthesize myelin components in the absence of neurons (Pfeiffer et al., 1993). Early reports from cultured rat OLs concluded that PLP was synthesized and incorporated into the PM without neurons (Hudson et al., 1989). Interestingly though, PM expression of PLP could not be detected for many days after intracellular pools of PLP were clearly detectable. The delayed PM expression of PLP raised the possibility that axonal signaling could speed up PM expression.The paper by Simons and colleagues in this issue demonstrates neuronal control of PLP trafficking (Trajkovic et al., 2006). Primary OLs, as well as two OL cell lines, contain PLP in a LE/L (as well as on the PM). This LE/L pool of PLP persists if neurons are absent from the culture. When OLs are cocultured with neurons, PLP is found with LE/L initially, but later disappears from there and increased amounts can be detected on the PM. When brain sections were costained against lysosomal markers and PLP, high colocalization of PLP with LE/L was detected in P7 mice while in P60 brains PLP did not colocalize with LE/L. Therefore, PLP localizes (at least partially) with LE/L in vivo and disappears from there upon myelination. This finding assuages much of the fear that PLP-containing LE/L are just a culture phenomenon or due to inappropriate expression levels (Kramer et al., 2001; Simons et al., 2002). The authors tested three explanations to account for their observations: increased proteolysis of PLP, decreased endocytosis, and/or increased exocytosis from LE/L. Proteolysis of PLP was found to be unaffected by neuronal coculture. Endocytosis (via a clathrin-independent, cholesterol-dependent, actin-dependent, and RhoA-dependent pathway), on the other hand, was decreased. Using PLP-GFP and lysotracker to mark LE/L in live OLs, the authors also found that the LE/L became much more mobile in the presence of neurons. To determine whether the moving LE/L in cocultured OLs can fuse with the PM and potentially deliver PLP sequestered in LE/L, the authors used total internal reflection fluorescence microscopy (TIRFM) on lysotracker-labeled OLs. Without neurons present, the LE/L was not found within 100 nm of the PM and was therefore invisible to TIRFM. When neurons were present, many LE/L were found near the PM and events suggestive of fusion could be observed at a rate of 1–2 events/min. Lastly, the authors determined that diffusible neuronal factors were sufficient to induce increased PLP surface expression. Addition of a membrane-permeable cAMP analogue to OLs in the absence of neurons led to increased PLP on the surface as well as high mobility of lysotracker pools containing PLP-GFP.These results suggest that diffusible neuronal factors (currently unknown) could activate cAMP signaling in OLs and regulate endocytosis and exocytosis of PLP. Exocytosis from LE/L is a regulated pathway in other cells as well (Blott and Griffiths, 2002). In OLs, at least some of the PLP could be stored in LE/L until neuronal promyelinating signals are received. Because many proteins arrive in the LE/L from the TGN, it would be interesting to investigate the potential neuronal regulation of PLP sorting events in the Golgi. Although we still await a complete quantitative account of what proportion of PLP is transported where and when, this paper presents an exciting advance in our understanding of the neuronal control of OL membrane traffic.